The proteasome is the central non-lysosomal protein degrading machinery in the cytoplasm and nucleus of all eukaryotic cells. It is in charge of degrading key proteins in metabolism, cell cycle control, and cell differentiation. Additionally, the proteasome is responsible for the generation of most peptide epitopes presented on MHC class I molecules (Fig. 1).
Fig.1: Antigen processing in the MHC class I-restricted pathway. Proteins that are synthesized in the cell (direct presentation) or are released from endosomes (cross-presentation) are polyubiquitylated in the cytoplasm and degraded by hybrid proteasomes consisting of the 20s proteasome core, the 19S regulator and PA28. The peptides that are produced are either of the ideal length for binding to MHC class I molecules (8–9 amino acids) or are amino-terminally extended precursors that can be further cleaved by aminopeptidases in the cytoplasm (such as leucine aminopeptidase, puromycin-sensitive aminopeptidase, bleomycin hydrolase and tripeptidyl peptidase II). Chaperones (such as heat shock protein 70 (HsP70), HsP90α and TriC) can stabilize the peptides in the cytoplasm to prevent their rapid degradation (for example by tripeptidyl peptidase II or thimet oligopeptidase). Transporter associated with antigen processing 1 (TAP1) and TAP2, which are attached to nascent MHC class I chains through tapasin, transport the peptides into the endoplasmic reticulum (ER), where they can be further trimmed at the N-terminus by er aminopeptidase 1 (ERAP1) and ERAP2. The oxidoreductase ERp57 ensures the maintenance of disulphide bridges in the MHC class I loading complex. Note that the carboxyl terminus of a peptide ligand for MHC class I molecules is mainly determined by proteasomal cleavage. The binding of peptides with high affinity to the MHC class I heavy chain–β2-microglobulin (β2m) complex induces a final folding and release of the MHC class I molecule from the ER lumenal chaperone calreticulin to allow exit from the ER and migration through the Golgi to the plasma membrane. TCR, T cell receptor. (Derived from Groettrup et al., Nat Rev Immunol. 2010;10(1):73-8.)
Exposure of cells to inflammatory cytokines leads to the replacement of the constitutive catalytic proteasome subunits β1c, β2c, and β5c by the inducible subunits LMP2 (β1i), MECL-1 (β2i), and LMP7 (β5i), respectively, during proteasome neosynthesis (Fig. 2).
Fig. 2: Subunit composition of the active sites of the constitutive proteasome and immunoproteasome. The proteolytic subunits of the constitutive proteasome are β1 (also known as PSMB6, Y, and δ), β2 (also known as PSMB7, Z, and MC14) and β5 (also known as PSMB5, X, MB1, and ε). The proteolytic immunoproteasome subunits are β1i (also known as PSMB9 and LMP2), β2i (also known as PSMB10, LMP10, and MECL-1) and β5i (also known as PSMB8 and LMP7). Compared with the constitutive proteasome, the immunoproteasome has a strongly decreased caspase-like activity and an increased chymotrypsin-like (CT-L) activity. (Derived from Groettrup et al., Nat Rev Immunol. 2010;10(1):73-8.)
The past two decades of immunoproteasome research have yielded a host of evidence that the immunoproteasome shapes antigen processing and presentation (Fig. 1) and by this means also the T cell repertoire (Basler et al., J Immunol. 2006;176(11):6665-72).
When we transferred T cells isolated from MECL-1-/-, LMP2-/-, and LMP7-/- mice into LCMV (lymphocytic choriomeningits virus) infected mice, we realized that the transferred cells were unable to survive in the recipient mice. We concluded that the immunoproteasome has a so far unrecognised function in expansion and survival of T cells. Therefore, we hypothesized that the immunoproteasome may qualify as a drug target for the silencing of undesired T cell responses e.g. in autoimmune diseases (Basler et al., J Immunol. 2006;176(11):6665-72/ Moebius et al., Eur J Immunol. 2010;40(12):3439-49.)
The pharmaceutical company Proteolix (later Onyx Pharmaceuticals, now Amgen) had developed the peptidomimetic inhibitor PR-957 (newly named ONX 0914), which selectively inhibited the chymotrypsin-like activity of LMP7. We could demonstrate that this LMP7-selective inhibitor blocked presentation of LMP7-specific, MHC-I-restricted antigens in vitro and in vivo. Furthermore, we could show that the immunoproteasome is also required for regulating the production of pro-inflammatory cytokines like IL-6, IFN-γ, TNF-α and IL-23 (Muchamuel et al., Nat Med., 2009;15(7):781-7). Additionally, selective inhibition or genetic ablation of β5i resulted in diminished Th1 and Th17 differentiation, enhanced development of regulatory T cells, but had no effect on Th2 differentiation (Fig. 3) (Muchamuel et al., Nat Med., 2009;15(7):781-7/Kalim et al., J Immunol., 2012;189(8):4182-93).
Fig. 3: Influence of LMP7 on T helper cell differentiation. Depending on the cytokine environment naive T helper cells (Th0) differentiate into Th1, Th2, Th17, or regulatory T cells (Treg). ↑: enhanced differentiation; →: no influence; ↓: reduced differentiation (Derived from Basler et al., Curr Opin Immunol., 2013;25(1):74-80.)
In mouse models of RA, inhibitor treatment reversed signs of disease and resulted in reductions in cellular infiltration, cytokine production and autoantibody levels. We also determined the effect of the inhibitor in cells derived from patients diagnosed with active RA. Inhibition of LMP7 blocked IL-23 production and TNF secretion in immune cells of these patients, suggesting that LMP7 regulates inflammatory cytokine production in cells from both normal healthy volunteers and patients with active RA (Muchamuel et al., Nat Med., 2009;15(7):781-7). Additionally, we investigated the effect of the LMP7-specific inhibitor in further models of autoimmune disorders. Thereby, the LMP7-specific inhibitor could dramatically reduce disease symptoms in a mouse colitis model (Basler et al., J Immunol., 2010;185(1):634-41., in a model for diabetes (Muchamuel et al., Nat Med., 2009;15(7):781-7), as well as in a mouse model for Multiple Sclerosis (Basler et al., EMBO Mol Med. 2014;6(2):226-38).
Recently, we have solved the crystal structures of the constitutive proteasome and immunoproteasome of the mouse at 2.9 Å resolution (Fig. 4) (Huber et al., Cell., 2012;148(4):727-38.). These data will promote the structure-guided design of inhibitory lead structures selective for immunoproteasomes.
Fig. 4: Overall architecture of the 28 subunits of the immunoproteasome drawn as spheres (A) and in ribbon representation (B).
The LMP7-specific inhibitor is the first proteasome inhibitor described that is selective for the CT-L subunit of the immunoproteasome and represents a powerful tool for understanding the role of LMP7 in immune responses. This study reveals a unique role for LMP7 in controlling pathogenic immune responses and provides a therapeutic rationale for targeting LMP7 in autoimmune disorders.
The mechanistic link between immunoproteasome activity and T helper cell function (Fig. 3) as well as cytokine production has remained elusive. We propose that the immunoproteasome might selectively processes a factor that is required for regulating cytokine production or T helper cell differentiation, but such a factor remains to be identified. Currently, we are investigating how the immunoproteasome plays a role in this processes on molecular levels.
- Basler M*, Mundt S*, Muchamuel T*, Moll C, Jiang J, Groettrup M, Kirk CJ. (2014) Inhibition of the immunoproteasome ameliorates experimental autoimmune encephalomyelitis. EMBO Mol Med 6(2):226-238. Pubmed
- Basler M, Kirk CJ, Groettrup M. (2013) The immunoproteasome in antigen processing and other immunological functions. Curr Opin Immunol, 2013;25(1):74-80. Pubmed
- Huber EM*, Basler M*, Schwab R, Kirk CJ, Heinemeyer W, Groettrup M, and Groll M.. (2012). Immuno- and Constitutive Proteasome Crystal Structures Reveal Differences in Substrate and Inhibitor Specificity. Cell: 148(4), 727-738. Pubmed
- Kalim KW, Basler M, Kirk CJ, Groettrup M. (2012). - Immunoproteasome subunit LMP7 deficiency and inhibition suppresses Th1 and Th17 but enhances regulatory T cell differentiation J Immunol 189(8):4182-4193 Pubmed
- Muchamuel T*, Basler M*, Aujay MA, Suzuki E, Kalim KW, Lauer C, Sylvain C, Ring ER, Shields J, Jiang J, Shwonek P, Parlati F, Demo SD, Bennett MK, Kirk CJ , and Groettrup M. (2009). - A selective inhibitor of the immunoproteasome subunit LMP7 blocks cytokine production and attenuates progression of experimental arthritis. - Nat. Med.: 15(7), 781-7. Pubmed
- Basler M, Moebius J, Elenich L, Groettrup M, and Monaco JJ. (2006). An altered T cell repertoire in MECL-1-deficient mice. J Immunol.: 176(11), 6665-6672. Pubmed
- Groettrup M, Kirk CJ, Basler M. (2010). - Proteasomes in immune cells: more than peptide producers? - Nature Rev. Immunol. 10(1):73-8. Pubmed
* shared first authorship